Download Determining the Role of Wnt Signaling in Zebrafish

Determining the Role of Wnt Signaling in Zebrafish Oocyte Maturation Through
Examination of β-catenin and Dishevelled mRNA Concentrations
Nathan Pincus* and Alyce DeMarais
Department of Biology, University of Puget Sound, Tacoma, WA 98416, [email protected]
Introduction
During oocyte maturation, the oocyte progresses from prophase I to
metaphase II of meiosis, and a multitude of other cellular changes occur1.
Wnt singaling pathways are known to regulate gene expression, cell behavior,
cell adhesion, and cell polarity, as well as play an essential role in embryonic
development2. Because of this, I am examining the role of Wnt signaling
pathways in the earlier process of oocyte maturation, specifically by looking
at two Wnt signaling pathway components: β-catenin (ctnnb1) and
Dishevelled (dvl2). β-catenin is a an interesting protein to study because it
plays a dual role as both a cell adhesion protein when attached to membranebound complexes, and a coactivator for transcription by the Wnt pathway
when free in the cytoplasm2, 3. Dishevelled is the “hub” of Wnt signaling and
plays a key role in relaying external signals to internal pathway components4.
Preliminary research has suggested that β-catenin increases in relative
cytoplasmic concentration after maturation, and my findings from last
summer showed that this change is not the result of migration from
cytoskeleton associated membrane-bound complexes5,. The first step in my
research is to determine a reference gene for zebrafish oocyte maturation, as
none are well classified for this specific scenario. I examined β –actin,
GAPDH and ef1-α, as these were found to have constant expression during
zebrafish embryo development or bovine oocyte development6, 7. This will be
followed by examining the changes in mRNA concentrations for β-catenin and
Dishevelled over the course of oocyte maturation in order to determine the
role and importance of the Wnt signaling pathway in this process. Changes in
mRNA concentrations are determined through real-time RTPCR analysis. The
results of my research will contribute to our understanding of the cellular
processes which occur during oocyte maturation, and the importance of
signaling pathways such as the Wnt pathway in these processes.
Results
Figure 1. Primer efficiency for actb1 and ctnnb1 at 56oC
Discussion
•All primers were successfully optimized for use in qPCR.
•Oocyte collection methods were redefined to include the
surrounding ovary tissue and immature oocytes as well as larger
oocytes. This was required to get sufficient RNA yields, and is
acceptable as the oocytes at this point do not produce their own
RNA, and instead are fed RNA by surrounding ovary cells.
• Gene expression analysis shows actb1 and ef1a as potential
reference genes with some complications from uneven
expression. Further testing will determine whether or not the
variance is acceptable.
•Gene expression analysis for ctnnb1 shows differential
expression for both control and test groups, with a spike in
expression at the four hour point. This raises the question of
whether spontaneous maturation is occurring in the control
group, or if there are other confounding factors.
Figure 2. Primer efficiency for dvl2 at 56oC
Materials and Methods
•Primer design and optimization
•Redefinition of oocyte collection procedure
•Induction of oocyte maturation with 1 μg/mL progesterone (DHP)
•Incubation of oocytes for 0, 1, 2, 4, 8, and 24 hours
•RNA extraction with Qiagen RNAeasy Mini Kit and conversion to cDNA
•Testing possible reference genes through qPCR
•actb1, gapdh, and ef1a
•Check for constant expression throughout all time-points
•Measuring expression of target genes ctnb1 and dvl2 with qPCR
•Run target gene against reference gene at all time-points.
•Determine gene expression relative to expression of the reference gene
Figure 3. Gene expression for actb1 and gapdh during maturation
Figure 4. Gene expression for actb1 and ef1a during
maturation
References
1.
2.
3.
4.
5.
6.
7.
Lessman CA: Oocyte maturation: Converting the zebrafish oocyte to the fertilizable egg. (2009) General and Comparative Endocrinology,
161, 53-57.
Moon R, Bowerman B, Boutros M, Perrimon N: The promise and perils of Wnt signaling through β-catenin. (2002) Science, 296, 1644-1646.
Ben-Ze’ev A, Shtutman M, Zhurinsky J: The integration of cell adhesion with gene expression: the role of β-catenin. (2000) Experimental Cell
Research, 261, 75-82.
Gao C, Chen Y: Dishevelled: The hub of Wnt signaling. (2010) Cellular Signalling, 22, 717-727
Hamilton K: Possible role of β catenin in oocyte maturation in zebrafish. (2006) unpublished manuscript.
Callard GV and McCurley AT: Characterization of housekeeping genes in zebrafish: male-female differences and effects of tissue type,
developmental state and chemical treatment. (2008) BMC Molecular Biology, 9, doi:10.1186/1471-2199/9/102.
Smith GW, Bettegowda A, Patel OV, and Ireland JJ: Quantitative analysis of messenger RNA abundance for ribosomal protein L-15,
cyclophilin-A, phosphoglycerokinase, β-glucuronidase, glyceraldehydes 3-phosphate dehydrogenase, β-actin, and histone H2A during
bovine oocyte maturation and early embryogenesis in vitro. (2006) Molecular Reproduction and Development, 73, 267-278.
Future Directions
•Continue work to define a reference gene for oocyte maturation,
focusing on actb1 and ef1a.
•Confirm if there are consistent and significant changes in ctnnb1
expression during oocyte maturation.
•Examine changes in dvl2 expression over time
•Examine the levels of Wnt signaling proteins during oocyte
maturation, potentially through Western blotting.
Acknowledgements
Figure 5. Gene expression for actb1 and ctnnb1
during maturation
I would like to thank the University of Puget Sound and the NASA
Washington Space Grant for funding my research, and Courtney
LaValle for teaching me to use the qPCR instrument and interpret
my results.